Establishment of the IL-17RA1-KO medaka strain
To KO the il17ra1, we deleted a genomic region spanning from exon 1 to 7 of the medaka il17ra1 (Fig. 1A). Three of the four designed crRNAs (crRNA1-2, crRNA7-1, and crRNA7-2) showed the high mutation efficiencies (Fig. S1). A mixture of these three crRNAs was injected back into medaka embryos to edit the genome. In this manner, we established IL-17RA1-KO medaka with approximately 5.4 kb of genome sequence deleted from exon 1 to 7. The deduced amino acid sequence of mutated IL-17RA1 lacked most parts of the extracellular domain (IL-17 fn III domain) predicted using the domain search tool, SMART 7 (http://smart.embl-heidelberg.de); the IL-17 fn III domain is important for binding IL-17 ligands (Fig. 1B). After microinjection, we obtained six mutant lines (KO line A–D, J, and K; Fig. S2A) with the same open-reading frame (ORF), as the 5¢ deletion occurred upstream of il17ra1 start codon. Specifically, the il17ra1 deletion started in the middle of the crRNA1-2 region (upstream of the original il17ra1 start codon), and exons 1 to 6 were completely lost in each case (Fig. S2B). At the amino acid level, the deletion corresponded to most of the extracellular IL-17 Fn III domain, with the downstream amino residues causing no codon frame shift (Fig. S2C). The ORF amplicon of WT il17ra1 was not amplified from the cDNA samples of IL-17RA1-KO medaka, whereas the remaining common region (1,491 bp) was amplified, and its sequence was confirmed by sequencing (Fig. S3A). Furthermore, qPCR analysis using a primer set designed against the common region of WT and mutated il17ra1 confirmed significantly lower expression in the posterior intestine of IL-17RA1-KO medaka (Fig. S3B).
Weight loss in IL-17RA1-KO medaka
The established IL-17RA1-KO medaka line was bred with heterozygous medaka, and genotyping by the HMA method was used to confirm each generation (Fig. S2D). It was impossible to maintain the IL-17RA1-KO medaka lines by mating homologous mutants. Homozygous individuals in all KO lines were lean, and the females did not lay eggs. Of the six IL-17RA1-KO lines, KO line C was chosen as the main line because its breeding and proliferation were more stable. Genotype comparisons were performed between WT and mutant (heterozygous and homozygous) populations immediately after hatching (at 0–1 day post-hatching [dph]) and at 110–120 dph. Furthermore, we performed a genotype comparison with our previously established IL-17A/F1-KO medaka line. No abnormalities were observed at 0–1 dph in IL-17RA1-KO medaka (Fig. 2A). However, at 110–120 dph, only the homologous mutants of IL-17RA1-KO presented significant decreases in body weight (Fig. 2B, C), which were also observed in all obtained homologous-mutant KO lines of IL-17RA, other than line C (i.e., lines A and K; Fig. S4). Furthermore, the proportion of surviving homologous mutants was significantly lower at 110–120 dph than at 0–1 dph (Fig. 2D).
Notably, we observed that the intestinal tract of IL-17RA1-KO medaka was significantly shorter, than that of the WT and IL-17A/F1-KO medaka (Fig. 3A, B). Histological observation of HE-stained sections revealed dramatically thinner muscle layers under the intestinal epithelium in IL-17RA-KO medaka than that in WT and IL-17A/F1-KO medaka (Fig. 3C).
Medaka anterior intestines expressed genes related to lipid-metabolism pathways, as observed in mammalian small intestines
We next performed RNA-seq analysis to investigate functional differences between the anterior and posterior intestines of medaka, and to assess the influences of IL-17RA1 KO on downstream gene-expression levels. cDNA libraries constructed with tissue sections from anterior and posterior medaka intestines are represented in Fig. 4A. After removing low-quality reads, adaptors, and reads with a high content of unknown bases, we obtained an average of 30,840,974 (anterior intestine/WT), 30,156,147 (posterior intestine/WT), 43,545,422 (anterior intestine/IL-17RA1-KO) and 38,596,287 (posterior intestine/IL-17RA1-KO) reads from the indicated transcriptome libraries. After annotation, 20,710 genes in the WT anterior intestine, 21,529 genes in the RA1-KO anterior intestine, 20,990 genes in the WT posterior intestine, and 21,734 genes in the RA1-KO posterior intestine were detected in each library (Fig. 4B). The overall gene-expression levels in the anterior and posterior intestines showed different patterns, which also differed from those of WT and IL-17RA1-KO medaka (Fig. 4C). In WT intestines, 139 genes showed higher expression levels in the anterior intestine than in the posterior intestine, and 174 genes showed significantly higher expression in the posterior intestine than in the anterior intestine. Tables S2 and S3 display the top 50 genes with large expression differences in the anterior and posterior intestines, respectively. Of the 139 genes up-regulated in the anterior intestine, 74 were also showed significantly higher expression in the anterior intestine of IL-17RA-KO medaka than in the posterior intestine of KO medaka. Similarly, of the 174 genes that were significantly up-regulated in WT posterior intestine, 98 genes were also expressed in IL-17RA-KO posterior intestine (Fig. S5A). GO analysis of the upregulated genes suggested that the anterior intestine produced various metabolites, such as lipids, organic acids, oxoacid, and carboxylic acid. In contrast, in the posterior intestine, genes related to proteolysis and cellular catabolic processes were significantly up-regulated (Fig. S5B). Furthermore, enrichment analysis of KEGG pathways showed that the anterior intestine exhibited an enhancement of the “fat digestion and absorption” pathway of the mammalian small intestine (Fig. 5A, B). Notably, marked up-regulation of the apolipoprotein A-I (apoa1b), group XIIB secretory phospholipase A2-like protein (pla2g12b), and monoacylglycerol O-acyltransferase 2 (mogat2) genes in both WT and IL-17RA-KO medaka was also confirmed by qPCR (Fig. 5C–E). In contrast, KEGG enrichment analysis showed that the posterior intestine exhibited enhanced expression of genes in the “lysosome” pathway (Fig. 6A, B). Of the genes with remarkably higher expression in the WT posterior intestine, up-regulation of cathepsin B (ctsbb), neuraminidase 1 (neu1), and mannosidase alpha class 2B member 1 (man2b1) was confirmed by qPCR (Fig. 6C–E).
IL-17RA1-KO medaka exhibited decreased IL-17 signalingand decreased expression of various metabolism- and immune-related genes
A comparison of IL-17RA1-KO and WT medaka intestines revealed 290 DEGs with significant changes (P < 0.05) in the anterior intestine (up-regulated genes: 167, down-regulated genes: 123). In the posterior intestine, 205 genes were identified as DEGs (up-regulated genes: 124, down-regulated genes: 81), as shown in Fig. 7A. Of these DEGs, genes down-regulated or up-regulated by 3-fold are shown in Tables S4–S7. We detected 24 DEGs commonly down-regulated in both the anterior and posterior intestines of IL-17RA1-KO, as well as 35 DEGs commonly up-regulated in both intestinal regions (Fig. 7B). We further visualized interactions between DEGs using the String App in Cytoscape. We examined potential relationships between IL-17RA and the DEGs by adding IL-17RA to the analysis and localized many metabolism-related genes; 47 genes clustered together with IL-17RA among the 123 down-regulated DEGs. Interestingly, IL-17C (an IL-17RA ligand) and the Th17 master transcription factor, nuclear receptor ROR-alpha (rorc), were included in this relationship. Furthermore, 10 genes including mevalonate pathway-related genes, mevalonate diphosphate decarboxylase (mvda), acetyl-CoA acetyltransferase 2 (acat2), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (hmgcs1), and 3-hydroxy-3-methylglutaryl-coenzyme A (hmgcr) showed particularly strong interactions (Fig. 8). These 47 genes are presented in Table 1.
Of the DEGs with increased expression in the anterior intestine, 102 genes formed a cluster containing IL-17RA. In particular, a series of collagen genes showed strong interactions (Fig. S6). Furthermore, among the DEGs that were significantly decreased in the posterior intestine, some were possibly related to IL-17RA1, including IL-17A/F1, growth arrest and DNA damage inducible alpha (gadd45a), chymotrypsin-like elastase family member 2A (ela2), neuraminidase 1 (neu1), and signal transducer and activator of transcription 3 (stat3) (Fig. S7A). However, we did not find a gene cluster among the elevated DEGs that interacted with IL-17RA (Fig. S7B).
Furthermore, GO analyses were performed on the DEGs using the DAVID program. Among the down-regulated DEGs in the anterior intestine of IL-17RA1-KO medaka, terms related to various metabolic process including lipid metabolism, steroid metabolism, lipid biosynthesis, and oxidation reduction were annotated in the biological process (BP) category. Of the immune terms, defense response (BP category) and cytokine binding (molecular function category) were also identified among the top 12 terms with the most hits (Fig. 9A). In contrast, among the down-regulated DEGs in the posterior intestine of IL-17RA1-KO, the most hits were obtained for proteolysis; hits were also obtained for lipid metabolism-related and oxidation-reduction terms, similar to those obtained for terms of the anterior intestine (Fig. 9B). Development of the circulatory and cardiovascular systems (Fig. S8A) were emphasized among the up-regulated DEGs in the anterior intestine of IL-17RA1-KO medaka, whereas oxidation-reduction process and organic acid metabolic process were the top two BP terms in the posterior intestine (Fig. S8B).
Among the down-regulated DEGs in the anterior intestine of IL-17RA1-KO medaka, KEGG pathway analysis revealed that four genes (mvda, acat2, hmgcs1, and hmgcr) were part of the mammalian mevalonate metabolic pathway (Fig. 10A). Down-regulated levels of mvda, acat2 and hmgcr were confirmed by qPCR analysis (Fig. 10B). The levels of IL-17 signaling-related genes were also confirmed by qPCR. RNA-seq analysis showed that the anterior intestine of IL-17RA1-KO, the master transcriptional factor of IL-17-producing lymphocytes (rorc) and il17c (an IL-17RA ligand). In posterior intestine of IL-17RA1-KO, il17a/f1 (an IL-17RA ligand) was significantly down-regulated. Conversely, stat3 shown to be down-regulated by RNA-seq analysis in the posterior intestine of IL-17RA-KO, did not show significant changes by qPCR analysis (Fig. 10C).
Comparison of RNA-seq results between IL-17A/F1-KO and IL-17RA1-KO medaka
Previously, we used IL-17A/F1, one of the ligands of IL-17RA knockout medaka (IL-17A/F1-KO) to reveal the role of IL-17 A/F signaling in the intestine and performed RNA-seq analysis of the whole intestinal tissues (GenBank accession number: DRA008715) . Comparing the down-regulated DEGs between IL-17A/F1-KO and IL-17RA1-KO medaka intestines showed that protein disulfide isomerase family A, member 2 (pdia2) and cytochrome P450 family 51 subfamily A, polypeptide 1 (cyp51) were significantly down-regulated in IL-17A/F1-KO and in both sections of IL-17RA1-KO medaka intestines. Furthermore, the anterior and posterior intestines of IL-17RA1-KO shared eight and seven down-regulated genes with IL-17A/F1-KO medaka, respectively (Table 2).